Transforming growth factor (TGF)-(1 plays a central role in the pathobiology of pulmonary fibrosis (PF), a debilitating and fatal illness that currently has no cure. TGF-(1 promotes PF by acting on both fibroblasts and epithelial cells, which are the two major cell types involved in the pathogenesis of PF. TGF-(1 initiates and sustains fibroblast activation and trans-differentiation into myofibroblasts, a hallmark of PF. In addition, TGF-(1 induces extracellular matrix (ECM) gene expression in myofibroblasts, the primary cellular source of ECM production in areas of active fibrogenesis. TGF-(1 induces growth arrest and apoptosis in alveolar type II (ATII) epithelial cells, thus preventing epithelial repair necessary for the resolution of PF. TGF-(1 also induces epithelial-mesenchymal transition in ATII cells, which has now been shown to occur in both human and animal models of PF. Our laboratory has made the sentinel observation that TGF-(1 induces Heparan sulfate (HS) 6- O-endosulfatase 1 and 2 (Sulf1 and Sulf2) in murine models of PF. Sulf1 and Sulf2 are the newly identified cell surface HS 6-O-endosulfatases, and have been shown to modulate HS-protein interactions by removing 6-O- sulfates from specific HS intra-chain sites. In vitro, TGF-(1 induces Sulf1 and Sulf2 in a cell-type specific manner, specifically Sulf1 in primary normal human lung fibroblasts and Sulf2 in A549 cells, an ATII cell line. Furthermore, blocking Sulf induction in lung fibroblasts and A549 cells altered their responses to TGF-(1. Finally, both Sulf1 and Sulf2 are up-regulated in human idiopathic pulmonary fibrosis. Based on our preliminary data, we hypothesize that heparan sulfate 6-O-endosulfatases, Sulf1 and Sulf2, modulate TGF-(1 function and the development of PF. In Aim 1, we will examine the role of Sulf1 in the development of PF. First, we will compare TGF-(1-induced PF in wild-type (WT) and Sulf1 knockout (KO) mice. Second, we will isolate primary lung fibroblasts from WT and Sulf1 KO mice and compare their fibrogenic properties in vitro. In Aim 2, we will examine the role of Sulf2 in the development of PF. First, we will compare TGF-(1-induced PF in WT and Sulf2 KO mice. Second, we will isolate primary ATII cells from WT and Sulf2 KO mice and compare their fibrogenic properties in vitro. In the in vivo studies, adenoviral vectors encoding active TGF-(1 or viral vector control will be used to induce PF in mice, and lung tissues will be harvested and examined at different time points following viral exposure for evidence of fibrosis and expression of fibrotic markers at both protein and mRNA levels using standard immunohistochemistry, in situ hybridization, Western Blotting and quantitative real-time RT-PCR. In the in vitro cell culture studies, cellular responses to TGF-(1 as well as other factors involved in PF will be examined including FGF and Wnt. To our knowledge, this is the first study to address the role of Sulf1 and Sulf2 in the development of PF. Our findings could lead to the development of new therapeutic interventions for PF as well as fibrogenic pathology in other organs. PUBLIC HEALTH RELEVANCE: There are currently over 200,000 patients in the United States and five million people worldwide suffering from pulmonary fibrosis (PF). Combining mouse genetics and in vitro cell culture studies, we will address in this study, for the first time, the role of Sulf1 and Sulf2 in the development of PF. Findings from this study may lead to the development of novel therapies for PF.